A recent estimate suggests roughly 70% of current lead compounds in modern drug discovery derive directly from the natural products, many of which are glycosylated bacterial metabolites. Potier, P. Actual. Chim. 11: 9 (1999). Thus, bacterial glycosyltransferases and their corresponding sugar substrates contribute significantly to the diversity of pharmaceutically important metabolites. A glycosylated metabolite is one that is comprised of both a central core structure (often called the “aglycon”) and various sugar (or “glycosyl”) attachments.
Carbohydrates are able to exhibit target specificity and often the affinity of carbohydrate ligands for their target are defined by the structure and length of the sugar chain carried by the aglycon. Traditionally, carbohydrate ligands of bioactive agents have been implicated in the control of drug pharmacokinetics such as absorption, distribution, metabolism and/or excretion. However, recent growing evidence has led to a change in this dogmatic view.
Pyran (or furan) ring rigidity in conjunction with glycosidic bond flexibility lends itself to preorganization while deoxygenated and/or functionalized sugars also provide unusual hydrophobic and hydrophilic domains. Furthermore, there exist many examples in which removal of these critical ligands leaves barren aglycons with little or no biological activity. Thus, carbohydrates provide great functional diversity to secondary metabolite activity. Thorson, J. S. et al. “Nature's Carbohydrate Chemists: The Enzymatic Glycosylation of Bioactive Bacterial Metabolites,” Curr. Org. Chem. 5: 139-167 (2001); Weymouth-Wilson, A. C. “The Role of Carbohydrates in Biologically Active Natural Products,” Nat. Prod. Rep. 14: 99-110 (1997).
Carbohydrate ligands often determine the specificity and affinity with which bioactive metabolites bind to DNA. One of the best characterized glycoconjugates is calicheamicin γ1I (FIG. 1, 1), a member of the enediyne family of antitumor antibiotics isolated from Micromonospora echinospora. Thorson et al “Enediyne Biosynthesis and Self Resistance: A Progress Report,” Bioorgan. Chem. 27: 172-188 (1999) and references therein; Thorson et al. “Understanding and Exploiting Nature's Chemical Arsenal: The Past, Present and Future of Calicheamicin Research,” Curr. Pharm. Des. 6: 1841-1879 (2000) and references therein. The aryltetrasaccharide of calicheamicin defines both the DNA binding specificity and the high affinity (estimated to be 106-108) of calicheamicin.
In the related enediyne neocarzinostatin (FIG. 1, 2), the carbohydrate ligand is 2,6-dideoxy-2-methylamino-α-D-galacto-hexopy-ranose 2-N-methyl-α-D-fucosamine) and, in contrast to most minor groove-binding aminoglycosyl ligands, the neocarzinostatin pyranose acts as an anchor, through numerous intermolecular contacts, and defines how deep neocarzinostatin can penetrate the major groove. This locks the molecule into position and thus, ultimately defines the specific sites of DNA-cleavage as well as enhances (possibly as an internal base) the efficiency of cleavage. Stassinopoulos et al. “Solution Structure of a Two-Base DNA Bulge Complexed with an Enediyne Cleaving Analog,” Science 272: 1943-1946 (1996); Myers et al. “A Comparison of DNA Cleavage by Neocarzinostatin Chromophore and Its Aglycon: Evaluating the Role of the Carbohydrate Residue,” J. Am. Chem. Soc. 119: 2965-2972 (1997).
Like the sugar ligands of calicheamicin γ1I and neocarzinostatin, the carbohydrate ligands of anthracyclines (e.g. daunorubicin, 5, among the most potent and widely used anticancer agents) are known to contribute directly to DNA binding, via intermolecular contacts, and to retard the activity of polymerases in some cases. Also, a direct correlation between increased glycosylation and lower toxicity has been demonstrated. Kirschning et al. “Chemical and Biochemical Aspects of Dexoysugars and Deoxysugar Oligosaccharides,” Top. Curr. Chem. 188: 1-84 (1997). Similar roles for the carbohydrates in DNA minor groove binding of the pluramycin antitumor antibiotics (e.g. altromycin B, a DNA alkylator, FIG. 1, 3), the antimicrobial aureolic acids (e.g. chromomycin A3, an inhibitor of replication/translation, FIG. 1, 8), and various other angucyclines, have been observed. Hansen et al. “Threads the DNA Helix Interacting with Both the Major and Minor Grooves to Position Itself for Site-Directed Alkylation of Guanine N7,” J. Am. Chem. Soc. 117: 2421-2429 (1995); Pavlopoulos et al. “Structural Characterization of the 1:1 Adduct Formed between the Antitumor Antibiotic Hedamycin and the Oligonucleotide Duplex d(CACGTG)2 by 2D NMR Spectroscopy.” Biochem. 35: 9314-9324 (1996); Pavlopoulos et al. “Characterization of the Sequential Non-covalent and Covalent Interactions of the Antitumor Antibiotic Hedamycin with Double Stranded DNA by NMR Spectroscopy,” J. Mol. Recognition 12: 346-354 (1999); Johnson et al. “Mechanisms and Pathways from Recent Deoxysugar Biosynthesis Research,” Curr. Opin. Chem. Biol. 2: 642-649 (1998); Keniry et al “The Three-Dimensional Structure of the 4:1 Mithramycin:d(ACCCGGGT)2 Complex: Evidence for an Interaction between the E Saccharides,” Biopolymers 54: 104-114 (2000).
Saccharides of secondary metabolites are also responsible for interaction with RNA. Examples include the orthosomycins such as the antibiotic evernimicin (FIG. 1, 11), which specifically binds to the 505 ribosomal subunits of E. coli and S. aureus and ultimately inhibits protein synthesis. McNicholas et al “Evernimicin Binds Exclusively to the 505 Ribosomal Subunit and Inhibits Translation in Cell-Free Systems Derived from both Gram-Positive and Gram-Negative Bacteria,” Antimicrob. Agents & Chemotherapy 44: 1121-1126 (2000).
Other examples include the macrolides (described further herein), such as erythromycin D (FIG. 2b, 18), which generally inhibit protein synthesis by inhibiting the 505 ribosome via carbohydrate ligand-mediated binding with the 23S ribosomal subunit and various proteins. Fish et al. “Structure-Activity Studies of Tylosin-related Macrolides,” J. Antibiot. 49: 1044-1048 (1996). Extensive work has established the critical importance of the macrolide carbohydrate ligands in bioactivity. Kurihara et al. “Analogues of Sixteen-Membered Macrolide Antibiotics. I. Synthesis of 4-O-Alkyl-L-cladinose Analogues via Glycosylation,” J. Antibiot. 49: 582-592 (1996). Likewise, the classical aminoglycosides, (e.g. streptomycin, FIG. 1, 6) interact with the small (30S) subunit of eubacteria-type ribosomes which generally leads to translational misreading.
Carbohydrate ligands also play a role in metabolites which interact with cell walls/membranes. For example, the non-ribosomal peptide antibiotic vancomycin (FIG. 1, 7) kills cells by binding to the N-acyl-D-Ala-D-Ala termini of uncrosslinked lipid-PP-disaccharide-pentape-peptides. Goldman et al., Curr. Med. Chem. 7: 801 (2000). While it is known that the carbohydrate portion of vancomycin is not directly involved in this binding event, deglycosylation or N-alkylation of the terminal vancosamine sugar of vancomycin shows remarkably different antibacterial profiles, while analogs with synthetically modified carbohydrates were found to operate via a mechanism distinct from that of vancomycin. Solenberg et al. “Production of Hybrid Glycopeptide Antibiotics in vitro and in Streptomyces toyocaensis,” Chem. Biol. 4: 195-202 (1997); Ge et al. “Reconstruction of Vancomycin by Chemical Glycosylation of the Pseudoaglycon,” J. Am. Chem. Soc. 120: 11014-11015 (1998); Thompson et al “Synthesis of Vancomycin from the Aglycon,” J. Am. Chem. Soc. 121: 1237 (1999); Ge et al. “Vancomycin Derivatives that Inhibit Peptidoglycan Biosynthesis without Binding D-Ala-D-Ala,” Science 284: 507-511 (1999).
As another example, the polyenes, such as amphotericin B (FIG. 1, 9), bind selectively to ergosterol in the cell membrane of susceptible fungi, inducing changes in permeability that ultimately lead to cell death. Georgopapadakou, “Antifungals: Mechanism of Action and Resistance, Established and Novel Drugs,” Curr. Opin. Microbiol. 1: 547-557 (1998); Abusalah, Brit. J. Biomed. Sci. 53: 122 (1996). In the amphotericin B-cholesterol aggregate cylindrical complex in the plasma membrane, critical hydrogen-bonding contacts between the polyene sugar and sterol contribute specificity for ergosterol over cholesterol.
Carbohydrate ligands often influence or determine interactions between bioactive metabolites and proteins. In this regard, the indolocarbazoles are an interesting class of metabolite. Prudhomme, Curr. Pharm. Des. 3: 265 (1997); Qu et al. “A DNA Binding Indolocarbazole Disaccharide Derivative Remains Highly Cytotoxic without Inhibiting Topoisomerase I,” Anti-Cancer Drug Des. 14: 433-442 (1999); Bailly et al. “Enhanced Binding to DNA and Topoisomerase I Inhibition by an Analog of the Antitumor Antibiotic Rebeccamycin Containing an Amino Sugar Residue,” Mol. Pharmacol. 55: 377-385 (1999); Bailly et al. “Recognition of Specific Sequences in DNA by a Topoisomerase I Inhibitor Derived from the Antitumor Drug Rebeccamycin,” Mol. Pharmacol. 53: 77-87 (1998); Goossens et al. “Cellular Uptake and Interaction with Purified Membranes of Rebeccamycin Derivatives,” Eur. J. Pharmacol. 389: 141-146 (2000). The indolocarbazoles, can be subdivided into two subgroups depending on the nature of the linkage between the carbohydrate residue and the heterocyclic chromophore. Compounds with the sugar attached to the two indole nitrogens (e.g. staurosporine, FIG. 1, 12) have little or no interaction with nucleic acids but strongly inhibit different protein kinases. In contrast, the second subgroup consists of indolocarbazole derivatives in which the carbohydrate moiety is attached to only one indole nitrogen, (e.g. rebeccamycin, 10) which does not inhibit PKC but instead its activity is attributed to the ability to induce topoisomerase-I-dependent DNA-strand breaks. These incredibly different activities attest to the critical role of the saccharide ligand.
As another example, novobiocin (FIGS. 1, 4, discussed further herein) is a naturally-occurring coumarin which targets DNA gyrase, the bacterial type II topoisomerase which can introduce negative supercoils into DNA using the free energy of ATP hydrolysis. Structural analyses reveal a significant overlap of the novobiocin sugar constituent and the binding site of the ATP adenine ring. Kampranis, S. C. et al. “Probing the Binding of Coumarins and Cyclothialidines to DNA Gyrase,” Biochem. 28: 1967-1976 (1999).
Macrolide antibiotics and coumarin antibiotics are clinically important examples of biologically active glycosylated secondary metabolites. The macrolides are a critical group of compounds due to their potent activity against Gram-positive bacteria. These compounds are generally classified by ring size of the aglycon lactone which contains either 12, 14, or 16 residues. Of these, the 14-membered ring and 16-membered ring families have been extensively studied from which erythromycin A1, oleandromycin, spiramycin, josamycin and midecamycin are used clinically. In general, these metabolites inhibit protein synthesis by inhibiting the 505 ribosome via specific binding with the 23S ribosomal subunit and various proteins. Fish, S. A. et al. (1996).
The 16-member macrolides are generally found to bind 23S rRNA and inhibit peptidyltransferase activity while the 14-member macrolides generally inhibit the translocation of peptidyl-tRNA. Extensive work has established the critical importance of the carbohydrate ligands in bioactivity. Weymouth-Wilson, A. C. (1997); Kurihara, K. et al. (1996); Bertho, G. et al. “Conformational Analysis of Ketolide, Conformations of RU 004 in Solution and Bound to Bacterial Ribosomes,” J. Med. Chem. 41: 3373-3386 (1998); Bertho, G. et al. “Solution Conformation of Methylated Macrolide Antibiotics Roxithromycin and Erythromycin Using NMR and Molecular Modeling. Ribosome-bound Conformation Determined by TRNOE and Formation of Cytochrome P450-metbolite Complex,” Internatl. J. Biol. Macromol. 22: 103-127 (1998); Bertho, G. et al. “Transferred Nuclear Overhauser Effect Study of Macrolide-Ribosome Interactions: Correlation between Antibiotic Activities and Bound Conformations,” Biorg. & Med. Chem. 6: 209-221 (1998); Gharbi-Benarous, J. et al. J. Chem. Soc. Per. Trans. II 529 (1999); Verdier, L. et al. Biorgan. & Med. Chem. 8: 1225 (2000).
Katz and coworkers have demonstrated the biosynthesis of the megalomicins (e.g. FIG. 2, 19) proceeds from erythronolide B (16) in a stepwise manner (FIG. 2b) and interestingly, the conversion of erythromycin D (18) to megalomycin A (19), via oxidation and the addition of a single sugar 2,3,4,6-tetradeoxy-3-dimethylamino-β-D-threo-hexopyranose (megosamine), changes the molecule's activity from an antibiotic (erythromycin D) to an antiparasitic/antiviral agent (megalomycin A). Volchegursky, Y. et al. “Biosynthesis of the Anti-Parasitic Agent Megalomicin: Transformation of Erythromycin to Megalomicin in Saccharopolyspora erythraea,” Mol. Microbiol. 37: 752-762 (2000).
Novobiocin (FIGS. 1, 4) is a naturally-occurring coumarin from Streptomyces spheroides which targets DNA gyrase. DNA gyrase from E. coli is an A2B2 complex in which each polypeptide displays distinct functional domains and the coumarins specifically inhibit the ATPase reaction of GyrB in a competitive manner. The complexes of the 24 kDa GyrB fragment with novobiocin and a related coumarin, chlorbiocin, show the binding sites for ATP and coumarins partially overlap. Tsai, F. T. F et al. Proteins 28: 41 (1997); Lewis, R. J. et al. EMBO J. 15: 1412 (1996). In particular, these high resolution structures reveal a significant overlap of the drug sugar constituent (3-β-aminocarbonyl)-6-deoxy-5-C-methyl-4-O-methyl-β-D-lyxo-hexopyranose, also known as β-D-noviose, in novobiocin) and the binding site of the ATP adenine ring with specific sugar-protein hydrogen-bonding interactions between the sugar C-2 and Asn 46, the sugar C-3 amide carbonyl with Thr 165 and amine with Asp 73/Val 43 main chain atoms. Site directed mutagenesis of these GyrB amino acids supports the structural assignments. Kampranis, S. C. et al. Biochem. 28: 1967 (1999). Interestingly, while these interactions are critical, the replacement of D-noviose with L-rhamnose has recently provided analogs with similar activity and potency. Ferroud, D. et al. Biorgan. & Med. Chem. Lett. 9: 2881 (1999). Furthermore, replacement of the C-3 acylamino substituent with reversed isosteres also provided highly potent analogs. Laurin, P. et al. Biorgan. & Med. Chem. Lett. 9: 2079 (1999). Recent studies also demonstrate a unique interaction of novobiocin with heat shock protein 90 (Hsp90), which shares homology with the a typical ATP-binding domaining of E. coli GyrB and stabilizes several oncogenic protein kinases. Marcu, M. G. J. Nat. Cancer Inst. 92: 242 (2000).
The gene cluster from S. spheroides which encodes for novobiocin biosynthesis and self resistance was recently cloned and a single glycosyltransferase gene (novM, accession AAF67506) was identified. Steffensky, M. et al. Antimicrob. Agents Chemotherap. 44: 1214 (2000). Given novobiocin contains a single saccharide, it is presumed novM encodes for the transfer of D-noviose from the activated dTDP-D-noviose to the aglycon novobiocic acid (FIG. 4, 20). The coumarins, while much more potent inhibitors of DNA gyrase in vitro than the clinically utilized quinolones, have failed clinically due to poor cell penetration, low solubility and toxicity in eukaryotes (perhaps due to this Hsp90 interaction). Thus, as an example of an area where engineering of secondary metabolites will be useful, glycosylated metabolites based on the coumarin aglycon but having altered carbohydrate moities may produce clinically useful compounds.
Both glycosyltransferases and nucleotidyltransferases play critical roles in the formation of glycosylated secondary metabolites. The first step in metabolite glycosylation is the reversible conversion of an α-D-hexose-1-phosphate to the corresponding nucleotide diphospho (NDP) hexose. Enzymes that catalyze this type of reaction (known as α-D-hexose-1-phosphate nucleotidyltransferases) are prevalent in nature and, regardless of their origins, are generally allosterically controlled with catalysis proceeding via an ordered bi-bi mechanism. Liu, H.-w. et al. “Pathways and Mechanisms in the Biogenesis of Novel Deoxysugars by Bacteria,” Annu. Rev. Microbiol. 48: 223-256 (1994).
The culminating attachment of a carbohydrate to a secondary metabolite aglycon (or growing saccharide chain) is catalyzed by the family of enzymes known as glycosyltransferases. These enzymes transfer a sugar, from its activated form (a nucleotide diphospho-sugar or NDP-sugar), to an acceptor nucleophile to form a glycosidic bond and NDP. These enzymes can catalyze transfer with retention (with respect to the NDP-sugar) or inversion of anomeric stereochemistry. Drawing from the glycosidase analogy, the current belief is “retaining” glycosyltransferases proceed via a double displacement mechanism, which utilizes an enzyme-glycoside covalent intermediate, while the “inverting” transferases proceed via a single displacement mechanism. Sinnott, M. L. “Mechanisms of Glycosyl Hydrolysis and Transfer,” Chem. Rev. 90: 1171-1202-1265 (1990). Based upon the known glycosylated metabolites, the majority of glycosyltransferases in secondary metabolism are “inverting” enzymes and the acceptor nucleophile is most often an aglycon or carbohydrate-derived heteroatom (O, N or S).
There are currently more than 70 putative secondary metabolite glycosyltransferase genes in the public database and these can be divided into three major families based upon sequence alignments. Thorson J. S. et al. (2001). Class I is the largest family and contains glycosyltransferases from both aromatic and macrolide metabolite pathways, Class II is predominately comprised of transferases associated with non-ribosomal peptides and glycolipids, while the majority of Class III enzymes are involved in metabolite inactivation. The number of known and putative secondary metabolite glycosyltransferase genes in the public database is growing rapidly, as this is an active area of research.
A number of genetic in vivo experiments have demonstrated that the glycosyltransferases of secondary metabolism (which include those for anthracyclines, angucyclines, nonribosomal peptides, macrolides and enediynes) are promiscuous with respect to the NDP-sugar donor. Thorson J. S. et al. (2001); Hutchinson, C. R. “Combinatorial Biosynthesis for New Drug Discovery,” Curr. Opin. Microbiol. 1: 319-329 (1998). While these in vivo experiments have provided novel metabolites, the newly formed metabolites, in most cases, were inactivated via host-catalyzed modification to prevent killing the host producing organism. Thus, in biosynthetically altering glycosylation, an in vitro scheme is desirable to eliminate this interference by host inactivation mechanisms.
The glycosyltransferases of secondary metabolism rely almost exclusively upon pyrimidine (uridine or thymidine) diphosphosugars, yet, in vitro studies in this area are severely lacking due to the inability to access the appropriate NDP-sugar substrates. Easy access to UDP- or dTDP-sugars would revolutionize the biochemical characterization and exploitation of these critical glycosyltransferases.
Surprisingly, a three dimensional structure for any enzyme from this important class of enzymes is lacking and of the many nucleotidyltransferases studied, the dTDP-α-D-glucose forming thymidylyltransferases have received the least attention. The best characterized thymidylyltransferase (rmlA-encoded Ep) is from Salmonella, which catalyzes the reaction shown in FIG. 2a. Lindquist, L. et al. “Purification, Characterization and HPLC Assay of Salmonella Glucose-1-phosphate Thymidylyltransferase from the Cloned rfbA Gene,” Eur. J. Biochem. 211: 763-770 (1993). Preliminary Ep substrate specificity studies, limited to only a few commercially available hexopyranosyl phosphates and NTPs, revealed Ep could utilize both dTTP and UTP as well as α-D-glucosamine-1-phosphate as a substitute for natural substrate (α-D-glucose-1-phosphate). Kinetic analysis revealed a ping-pong mechanism with K.sub.m values for the forward direction for dTTP and α-D-glucose-1-phosphate of 0.02 mM and 0.11 mM, respectively. In the reverse reaction the Km values for dTDP-α-D-glucose and diphosphate were 0.083 mM and 0.15 mM, respectively. Lindquist, L. et al. (1993).
The above examples illustrate that carbohydrate ligands often define the biological activity of a particular secondary metabolite and suggest alteration of saccharide ligands should lead to new compounds which may display novel biological activity. However, the complex structure of most glycosylated natural products preclude the ability to synthetically exchange their sugar ligands.
Further, while in vivo experiments have provided novel metabolites, the newly formed metabolites, in most cases, were inactivated via host-catalyzed modification to prevent killing the host producing organism. As the organisms producing the novel metabolites are killed, it is not feasible to produce sufficient amounts of novel metabolites for analysis or therapeutic use in in vivo systems. Additionally, producing novel metabolites in vivo requires the use of recombinant DNA technology to alter gene expression. Such methods are too time consuming for rapid production of numerous novel metabolites for testing as drug candidates. Further still, the production of these new agents was also severely limited by the host's biosynthetic machinery so that the number and diversity of compounds that may be produced by such methods is likewise severely limited.
Thus, for biosynthetically altering glycosylation, an in vitro scheme is needed to eliminate the problems associated with in vivo manipulation. Further, a scheme that allows such manipulation despite the complexities of biologically active secondary metabolites is needed.